Symposium_Gerlai_Willemsen by girlbanks


									           Zebrafish as a new model organism for Parkinson’s disease
                       Rob Willemsen, Wiebren Hasselaar, Herma van der Linde, Vincenzo Bonifati
                 Dept. of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands,

Parkinson’s Disease                                                           development (using morpholino gene knockdown techniques)
                                                                              creates easy access to new animal models. Although the
Parkinson’s Disease (PD) is the second most common human
                                                                              zebrafish brain does not contain a mesencephalic region
neurodegenerative disorder. PD patients present aberrant
                                                                              comparable to the substantia nigra, treatment with MPTP (a
motor activity with resting tremor, muscular rigidity,
                                                                              PD-inducing drug in humans, apes and to a lesser extent mice)
bradykinesia and postural imbalance [1, 2]. The cause of these
                                                                              showed a direct effect on diencephalic dopaminergic neurons.
symptoms is loss of dopaminergic neurons in the substantia
                                                                              This resulted in a loss of diencephalic dopaminergic neurons
nigra, pars compacta. Another pathological hallmark of PD is
                                                                              and an aberrant swimming pattern, illustrating that zebrafish
the frequent formation of Lewy bodies in surviving neurons.
                                                                              can develop a phenotype comparable to PD [8]. Importantly,
Lewy bodies are cytoplasmic aggregates of insoluble proteins.
                                                                              the homologues of the two selected PD-causing genes have
About 85-90% of the PD cases are sporadic with a complex
                                                                              been identified in zebrafish and morpholinos to study gene
aetiology. In 10-15% of the PD patients, a familial
                                                                              knockdown are available.
predisposition can be found. Linkage analysis in these families
conclusively identified -synuclein, parkin, PINK1, DJ-1,
LRRK2 and ATP13A2 as PD-causing genes [1, 3, 4]. The                          Aims
normal cellular function of these genes suggests that several                 Our research project aims at accomplishing two objectives.
pathways can be involved in neurodegeneration, including                      The first objective is a fundamental research question:
oxidative stress, proteosomal and mitochondrial dysfunction                   Understanding the cellular function of atp13a2 and lrrk2
and protein aggregation and misfolding [5]. Our research is                   during brain development and in adult brain.
focused on two PD-causing genes, that is, LRRK2 and
ATP13A2. LRRK2 has been implicated in both familial and                       The second objective is of translational nature: understanding
sporadic cases but the precise role of the LRRK2 protein in                   the molecular mechanisms underlying PD by generating
neurodegeneration is not clear. Clinical relevance is                         animal models of PD using zebrafish.
implicated by the similarity between the PD-phenotype of
patients carrying mutations in this gene and that of sporadic                 Methods
PD patients. The relatively high mutation rate in sporadic PD                 Manipulation of gene expression in zebrafish includes: gene
in some populations also adds to the hypothesis that LRRK2                    knockdown and transgenic strategies. For gene knockdown,
plays a key role in PD aetiology [6]. The other gene,                         we will use antisense oligonucleotides with a synthetic
ATP13A2, was identified in Kufor-Rakeb Syndrome. Patients                     backbone called morpholinos (MOs). MOs are 25 basepairs
are bound to bed before the age of 15 due to severe Levodopa-                 long and are designed to uniquely bind to our gene of interest,
responsive Parkinsonism and pyramidal symptoms. This gene                     thereby disrupting translation initiation or pre-mRNA splicing
was selected because of its severe phenotype and autosomal                    by binding to the pre-mRNA. MOs are used for functional
recessive inheritance. In the human patients these mutations                  genomic applications and have been shown to be very
result in a loss of functional protein. The co-localization of                effective in zebrafish. MOs are injected in the yolk sac of 1 or
ATP13A2 and -Synuclein [7] in lysosomal inclusions also                       2 cells-stage embryos and knockdown of gene function is
suggest a role for this gene in Lewy Body formation.                          transient (till 5 days post fertilization=dpf). For the generation
                                                                              of transgenic zebrafish, we will microinject plasmid DNA,
Zebrafish                                                                     containing our gene of interest fused to EGFP under the
Recently we have implemented a new simple vertebrate model                    control of a brain-specific promoter (GATA-2 or Tyrosine
with a richness of embryology/anatomy and genetics                            Hydroxylase (TH) promoter) into the cytoplasm of a 1-cell
knowledges, creating a new vertebrate model system for PD,                    stage embryo.
the zebrafish (Danio rerio).
                                                                              Both knockdown morphants and transgenic zebrafish will be
The zebrafish, a fresh water tropical fish, is a premiere model               further characterized for the presence of a phenotype,
organism to study vertebrate development. Fast external                       including microscopical screening for morphological features,
development and transparency during embryogenesis allow                       in situ hybridization (ISH) strategies using molecular probes
for visual screening at the macroscopical and microscopical                   to study gene expression patterns, immunohistochemical
level, including visualization of organogenesis. High fecundity               techniques (i.e. TH expression) and biochemical analyses
and short generation times facilitate genetic analyses.                       (Western blot). Another important aspect of characterization
Importantly, The Sanger Institute has recently released                       of a phenotype is the use of behavioural tests. Recently, a
assembly version Zv7, an almost finished sequence, of the                     system to monitor locomotor behaviour in zebrafish has been
1.5Gb zebrafish genome. Zebrafish may be a particular                         established (Noldus Information Technology bv; EthoVision).
powerful model for the study of human disease because many                    This system enables to record movement and swimming
cellular processes are conserved throughout vertebrate                        patterns of zebrafish embryos (6 dpf) in a highthroughput
evolution, including the corresponding disease genes. Finally,                fashion. Currently, this system is implemented at Erasmus
the ability to generate                                                       MC. Activity is monitored by a camera and the software
transgenic zebrafish allows for (over-) expression as well as                 detects the fish. This data visualizes the swimming pattern and
for the suppression of gene expression during early                           allows for statistical analysis of locomotor activity (figure 1).

                                                                 Proceedings of Measuring Behavior 2008 (Maastricht, The Netherlands, August 26-29, 2008)
50                                     Eds. A.J. Spink, M.R. Ballintijn, N.D. Bogers, F. Grieco, L.W.S. Loijens, L.P.J.J. Noldus, G. Smit, and P.H. Zimmerman
                                                                                        2.    Twelves, D., K.S. Perkins, and C. Counsell. (2003) Systematic
                                                                                              review of incidence studies of Parkinson's disease. Mov Disord
                                                                                              18(1), 19-31.
                                                                                        3.    Gasser, T. (2005) Genetics of Parkinson's disease. Curr Opin
                                                                                              Neurol. 18(4), 363-9.
                                                                                        4.    Ramirez, A., et al. (2006) Hereditary parkinsonism with
                                                                                              dementia is caused by mutations in ATP13A2, encoding a
                                                                                              lysosomal type 5 P-type ATPase. Nat Genet 38(10), 1184-91.
                                                                                        5.    Wood-Kaczmar, A., S. Gandhi, and N.W. Wood (2006)
                                                                                              Understanding the molecular causes of Parkinson's disease.
                                                                                              Trends Mol Med. 12(11), 521-8.
                                                                                        6.    Bonifati, V. (2007) LRRK2 low-penetrance mutations
     Figure 1. Zoom in on 2 wells of a 96 well plate with plots of the                        (Gly2019Ser) and risk alleles (Gly2385Arg)-linking familial and
     swimming pattern of larvae at 6dpf during 5 minutes. The larva in                        sporadic Parkinson's disease. Neurochem Res 32(10), 1700-8.
     the left well is treated with 10 M Diazepam, while the larva on                    7.    Wei, J., et al. (2007) Enhanced lysosomal pathology caused by
     the right is an untreated control                                                        beta-synuclein mutants linked to dementia with Lewy bodies. J
                                                                                              Biol Chem. 282(39), 28904-14.
                                                                                        8.    Panula, P., et al. (2006) Modulatory Neurotransmitter Systems
                                                                                              and Behavior: Towards Zebrafish Models of Neurodegenerative
References                                                                                    Diseases. Zebrafish 3(2), 235-47.
1.      Hardy, J., et al. (2006) Genetics of Parkinson's disease and
        parkinsonism. Ann Neurol 60(4), 389-98.

Proceedings of Measuring Behavior 2008 (Maastricht, The Netherlands, August 26-29, 2008)
Eds. A.J. Spink, M.R. Ballintijn, N.D. Bogers, F. Grieco, L.W.S. Loijens, L.P.J.J. Noldus, G. Smit, and P.H. Zimmerman                                    51

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